Recombinant DNA and Biotechnology

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					Recombinant DNA and
   Biotechnology
16                   Recombinant DNA and Biotechnology

     • Cleaving and Rejoining DNA
     • Getting New Genes into Cells
     • Sources of Genes for Cloning
     • Some Additional Tools for DNA Manipulation
     • Biotechnology: Applications of DNA Manipulation
16                              Cleaving and Rejoining DNA

     • Recombinant DNA technology is the
       manipulation and combination of DNA molecules
       from different sources.
     • Recombinant DNA technology uses the
       techniques of sequencing, rejoining, amplifying,
       and locating DNA fragments, making use of
       complementary base pairing.
16                                Cleaving and Rejoining DNA

     • Bacteria defend themselves against invasion by
       viruses by producing restriction enzymes which
       catalyze the cleavage of DNA into small fragments.
     • The enzymes cut the bonds between the 3
       hydroxyl of one nucleotide, and the 5 phosphate of
       the next.
     • There are many such enzymes, each of which
       recognizes and cuts a specific sequence of bases,
       called a recognition sequence or restriction site
       (4 to 6 base pairs long).
Figure 16.1 Bacteria Fight Invading Viruses with Restriction Enzymes
16                               Cleaving and Rejoining DNA

     • Host DNA is not damaged due to methylation of
       certain bases at the restriction sites; this is
       performed by enzymes called methylases.
     • The enzyme EcoRI cuts DNA with the following
       paired sequence:
         5 ... GAATTC ... 3
         3... CTTAAG ... 5
     • Notice that the sequence is palindromic: It reads
       the same in the 5-to-3 direction on both strands.
16                              Cleaving and Rejoining DNA

     • Using EcoRI on a long stretch of DNA would
       create fragments with an average length of 4,098
       bases.
     • Using EcoRI to cut up small viral genomes may
       result in only a few fragments.
     • For a eukaryotic genome with tens of millions of
       base pairs, the number of fragments will be very
       large.
     • Hundreds of restriction enzymes have been
       purified from various organisms, and these
       enzymes serve as ―knives‖ for genetic surgery.
16                              Cleaving and Rejoining DNA

     • The fragments of DNA can be separated using gel
       electrophoresis. Because of its phosphate groups,
       DNA is negatively charged at neutral pH.
     • When DNA is placed in a semisolid gel and an
       electric field is applied, the DNA molecules
       migrate toward the positive pole.
     • Smaller molecules can migrate more quickly
       through the porous gel than larger ones.
     • After a fixed time, the separated molecules can
       then be stained with a fluorescent dye and
       examined under ultraviolet light.
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)
16                              Cleaving and Rejoining DNA

     • Electrophoresis gives two types of information:
         Size of the DNA fragments can be determined
          by comparison to DNA fragments of known
          size added to the gel as a reference.
         A specific DNA sequence can be determined
          by using a complementary labeled single-
          stranded DNA probe.
     • The specific fragment can be cut out as a lump of
       gel and removed by diffusion into a small volume
       of water.
Figure 16.3 Analyzing DNA Fragments
16                                Cleaving and Rejoining DNA

     • Some restriction enzymes cut DNA strands and
       leave staggered ends of single-stranded DNA, or
       ―sticky‖ ends, that attract complementary
       sequences.
     • If two different DNAs are cut so each has sticky
       ends, fragments with complementary sticky ends
       can be recombined and sealed with the enzyme
       DNA ligase.
     • These simple techniques, which give scientists the
       power to manipulate genetic material, have
       revolutionized biological science in the past 30
       years.
Figure 16.4 Cutting and Splicing DNA
16                             Getting New Genes into Cells

     • The goal of recombinant DNA work is to produce
       many copies (clones) of a particular gene.
     • To make protein, the genes must be introduced,
       or transfected, into a host cell.
     • The host cells or organisms, referred to as
       transgenic, are transfected with DNA under
       special conditions.
     • The cells that get the DNA are distinguished from
       those that do not by means of genetic markers,
       called reporter genes.
16                            Getting New Genes into Cells

     • Bacteria have been useful as hosts for
       recombinant DNA.
          Bacteria are easy to manipulate, and they
           grow and divide quickly.
          They have genetic markers that make it easy
           to select or screen for insertion.
          They have been intensely studied and much of
           their molecular biology is known.
16                            Getting New Genes into Cells

     • Bacteria have some disadvantages as well.
         Bacteria lack splicing machinery to excise
          introns.
         Protein modifications, such as glycosylation
          and phosphorylation, fail to occur as they
          would in a eukaryotic cell.
         In some applications, the expression of the
          new gene in a eukaryote (the creation of a
          transgenic organism) is the desired outcome.
16                             Getting New Genes into Cells

     • Saccharomyces, baker’s and brewer’s yeast, are
       commonly used eukaryotic hosts for recombinant
       DNA studies.
     • In comparison to many other eukaryotic cells,
       yeasts divide quickly, they are easy to grow, and
       have relatively small genomes (about 20 million
       base pairs).
16                               Getting New Genes into Cells

     • Plants are also used as hosts if the goal is to
       make a transgenic plant.
     • It is relatively easy to regenerate an entire plant
       from differentiated plant cells because of plant cell
       totipotency.
     • The transgenic plant can then reproduce naturally
       in the field and will carry and express the gene on
       the recombinant DNA.
16                             Getting New Genes into Cells

     • New DNA can be introduced into the cell’s
       genome by integration into a chromosome of the
       host cell.
     • If the new DNA is to be replicated, it must become
       part of a segment of DNA that contains an origin
       of replication called a replicon, or replication
       unit.
16                             Getting New Genes into Cells

 • New DNA can be incorporated into the host cell by a
   vector, which should have four characteristics:
      The ability to replicate independently in the host
       cell
      A recognition sequence for a restriction enzyme,
       permitting it to form recombinant DNA
      A reporter gene that will announce its presence in
       the host cell
      A small size in comparison to host chromosomes
16                                 Getting New Genes into Cells

     • Plasmids are ideal vectors for the introduction of
       recombinant DNA into bacteria.
     • A plasmid is small and can divide separately from
       the host’s chromosome.
     • They often have just one restriction site, if any, for a
       given restriction enzyme.
     • Cutting the plasmid at one site makes it a linear
       molecule with sticky ends.
     • If another DNA is cut with the same enzyme, it is
       possible to insert the DNA into the plasmid.
     • Plasmids often contain antibiotic resistance genes,
       which serve as genetic markers.
Figure 16.5 (a) Vectors for Carrying DNA into Cells
16                                Getting New Genes into Cells

     • Only about 10,000 base pairs can be inserted into
       plasmid DNA, so for most eukaryotic genes a vector
       that accommodates larger DNA inserts is needed.
     • For inserting larger DNA sequences, viruses are
       often used as vectors.
     • If the genes that cause death and lysis in E. coli are
       eliminated, the bacteriophage l can still infect the
       host and inject its DNA.
     • The deleted 20,000 base pairs can be replaced by
       DNA from another organism, creating recombinant
       viral DNA.
16                              Getting New Genes into Cells

     • Bacterial plasmids are not good vectors for yeast
       hosts because prokaryotic and eukaryotic DNA
       sequences use different origins of replication.
     • A yeast artificial chromosome, or YAC, has
       been made that has a yeast origin of replication, a
       centromere sequence, and telomeres, making it a
       true eukaryotic chromosome.
     • YACs have been engineered to include
       specialized single restriction sites and selectable
       markers.
     • YACs can accommodate up to 1.5 million base
       pairs of inserted DNA.
Figure 16.5 (b) Vectors for Carrying DNA into Cells
16                               Getting New Genes into Cells

     • Plasmid vectors for plants include a plasmid found
       in the Agrobacterium tumefaciens bacterium,
       which causes the tumor-producing disease, crown
       gall, in plants.
     • Part of the tumor-inducing (Ti) plasmid of A.
       tumefaciens is T DNA, a transposon, which
       inserts copies of itself into the host chromosomes.
     • If T DNA is replaced with the new DNA, the
       plasmid no longer produces tumors, but the
       transposon still can be inserted into the host cell’s
       chromosomes.
     • The plant cells containing the new DNA can be
       used to generate transgenic plants.
Figure 16.5 (c) Vectors for Carrying DNA into Cells
16                              Getting New Genes into Cells

     • When a population of host cells is treated to
       introduce DNA, just a fraction actually incorporate
       and express it.
     • In addition, only a few vectors that move into cells
       actually contain the new DNA sequence.
     • Therefore, a method for selecting for transfected
       cells and screening for inserts is needed.
     • A commonly used approach to this problem is
       illustrated using E. coli as hosts, and a plasmid
       vector with genes for resistance to two antibiotics.
Figure 16.6 Marking Recombinant DNA by Inactivating a Gene
16                                Getting New Genes into Cells

     • Other methods have since been developed for
       screening.
     • The gene for luciferase, the enzyme that makes
       fireflies glow in the dark, has been used as a
       reporter gene.
     • Green fluorescent protein, which is the product of a
       jellyfish gene, glows without any required substrate.
     • Cells with this gene in the plasmid grow on
       ampicillin and glow when exposed to ultraviolet
       light.
16                            Sources of Genes for Cloning

     • Gene libraries contain fragments of DNA from an
       organism’s genome.
     • Restriction enzymes are used to break
       chromosomes into fragments, which are inserted
       into vectors and taken up by host cells.
Figure 16.7 Construction of a Gene Library
16                             Sources of Genes for Cloning

     • Using plasmids for insertion of DNA, about one
       million separate fragments are required for the
       human genome library.
     • Phage l, which carries four times as much DNA
       as a plasmid, is used to hold these random
       fragments.
     • It takes about 250,000 different phage to ensure a
       copy of every sequence.
     • This number seems large, but just one growth
       plate can hold as many as 80,000 phage colonies.
16                              Sources of Genes for Cloning

     • A smaller DNA library can be made from
       complementary DNA (cDNA).
     • Oligo dT primer is added to mRNA tissue where it
       hybridizes with the poly A tail of the mRNA
       molecule.
     • Reverse transcriptase, an enzyme that uses an
       RNA template to synthesize a DNA–RNA hybrid, is
       then added.
     • The resulting DNA is complementary to the RNA
       and is called cDNA. DNA polymerase can be used
       to synthesize a DNA strand that is complementary
       to the cDNA.
Figure 16.8 Synthesizing Complementary DNA
16                             Sources of Genes for Cloning

     • If the amino acid sequence of a protein is known,
       it is possible to synthesize a DNA that can code
       for the protein.
     • Using the knowledge of the genetic code and
       known amino acid sequences, the most likely
       base sequence for the gene may be found.
     • Often sequences are added to this sequence to
       promote expression of the protein.
     • Human insulin has been manufactured using this
       approach.
16                             Sources of Genes for Cloning

     • With synthetic DNA, mutations can be created
       and studied.
     • Additions, deletions, and base-pair substitutions
       can be manipulated and tracked.
     • The functional importance of certain amino acid
       sequences can be studied.
     • The signals that mark proteins for passage
       through the ER membrane were discovered by
       site-directed mutagenesis.
16              Some Additional Tools for DNA Manipulation

     • Homologous recombination is used to study the
       role of a gene at the level of the organism.
     • In a knockout experiment, a gene inside a cell is
       replaced with an inactivated gene to determine the
       inactivated gene’s effect.
     • This technique is important in determining the roles
       of genes during development.
Figure 16.9 Making a Knockout Mouse (Part 1)
Figure 16.9 Making a Knockout Mouse (Part 2)
16            Some Additional Tools for DNA Manipulation

     • The emerging science of genomics has to
       contend with two difficulties:
         The large number of genes in eukaryotic
          genomes
         The distinctive pattern of gene expression in
          different tissues at different times
     • To find these patterns, DNA sequences have to
       be arranged in an array on some solid support.
     • DNA chip technology provides these large arrays
       of sequences for hybridization.
Figure 16.10 DNA on a Chip
16             Some Additional Tools for DNA Manipulation

     • Analysis of cellular mRNA using DNA chips:
         In a process called RT-PCR, cellular mRNA is
          isolated and incubated with reverse
          transcriptase (RT) to make complementary
          DNA (cDNA). The cDNA is amplified by PCR
          prior to hybridization.
         The amplified cDNA is coupled to a
          fluorescent dye and then hybridized to the
          chip.
         A scanner detects glowing spots on the array.
          The combinations of these spots differ with
          different types of cells or different physiological
          states.
16             Some Additional Tools for DNA Manipulation

     • DNA chip technology can be used to detect
       genetic variants and to diagnose human genetic
       diseases.
     • Instead of sequencing the entire gene, it is
       possible to make a chip with 20-nucleotide
       fragments including every possible mutant
       sequence.
     • Hybridizing that sequence with a person’s DNA
       may reveal whether any of the DNA hybridized to
       a mutant sequence on the chip.
16            Some Additional Tools for DNA Manipulation

     • Base-pairing rules can also be used to stop
       mRNA translation.
     • Antisense RNA is complementary to a sequence
       of mRNA.
     • The antisense RNA forms a double-stranded
       hybrid with an mRNA, which inhibits translation.
     • These hybrids are broken down rapidly in the
       cytoplasm, so translation does not occur.
     • In the laboratory, antisense RNA can be made
       and added to cells to block translation.
Figure 16.11 Using Antisense RNA and RNAi to Block Translation of mRNA
16          Some Additional Tools for DNA Manipulation

 • A related technique uses interference RNA (RNAi)
   which inhibits mRNA translation in the inactive X
   chromosome of mammals.
 • Scientists can synthesize a small interfering RNA
   (siRNA) to inhibit translation of any known gene.
16             Some Additional Tools for DNA Manipulation

     • The two-hybrid system allows scientists to test
       for protein interactions within a living cell.
     • A two-hybrid system uses a transcription factor
       that activates the transcription of an easily
       detectable reporter gene.
     • This transcription factor has two domains: one
       that binds to DNA at the promoter, and another
       that binds to the transcription complex to activate
       transcription.
     • An example is the yeast two-hybrid system.
Figure 16.12 The Two-Hybrid System
16       Biotechnology: Applications of DNA Manipulation

     • Biotechnology is the use of microbial, plant, and
       animal cells to produce materials—such as foods,
       medicines, and chemicals—that are useful to
       people.
     • The use of yeast to create beer and wine and
       bacterial cultures to make yogurt and cheese are
       examples of centuries-old biotechnology.
     • Gene cloning techniques of modern molecular
       biology have vastly increased the number of these
       products beyond those that are naturally made by
       microbes.
16       Biotechnology: Applications of DNA Manipulation

     • Expression vectors are typical vectors, but they
       also have extra sequences needed for the foreign
       gene to be expressed in the host cell.
     • An expression vector might have an inducible
       promoter, which can be stimulated into expression
       by responding to a specific signal such as a
       hormone.
     • A tissue-specific promoter is expressed only in a
       certain tissue at a certain time.
     • Targeting sequences are sometimes added to
       direct the protein product to an appropriate
       destination.
Figure 16.13 An Expression Vector Allows a Foreign Gene to Be Expressed in a Host Cell
16       Biotechnology: Applications of DNA Manipulation

     • Many medical products have been made using
       recombinant DNA technology.
     • For example, tissue plasminogen activator (TPA),
       is currently being produced in E. coli by
       recombinant DNA techniques.
     • TPA is an enzyme that converts blood
       plasminogen into plasmin, a protein that dissolves
       clots.
     • Recombinant DNA technology has made it
       possible to produce the naturally occurring protein
       in quantities large enough to be medically useful.
Figure 16.14 Tissue Plasminogen Activator: From Protein to Gene to Drug
Table 16.1 Some Medically Useful Products of Biotechnology
16         Biotechnology: Applications of DNA Manipulation

     • Selective breeding has been used for centuries to
       improve plant and animal species to meet human
       needs.
     • Molecular biology is accelerating progress in these
       applications.
     • There are three major advantages over traditional
       techniques:
         Specific genes can be affected.
         Genes can be introduced from other organisms.
         Plants can be regenerated much more quickly
          by cloning than by traditional breeding.
16       Biotechnology: Applications of DNA Manipulation

     • Insecticides tend to be nonspecific, killing both
       pest and beneficial insects. They can also be
       blown or washed away to contaminate and pollute
       non-target sites.
     • Bacillus thuringiensis bacteria produce a protein
       toxin that kills insect larvae pests and is 80,000
       times more toxic than the typical chemical
       insecticide.
     • Transgenic tomato, corn, potato, and cotton
       plants have been made that produce a toxin from
       B. thuringiensis.
16       Biotechnology: Applications of DNA Manipulation

     • The process of producing pharmaceuticals using
       agriculture is nicknamed ―pharming.‖
     • Transgenic sheep are being used to produce
       human a-1-antitrypsin (a-1-AT) in their milk; this
       protein inhibits the enzyme elastase, which
       breaks down connective tissue in the lungs.
       Treatment with a-1-AT alleviates symptoms in
       people suffering from emphysema.
     • Other products of ―pharming‖ include blood
       clotting factors and antibodies for treating colon
       cancer.
16       Biotechnology: Applications of DNA Manipulation

     • Crops that are resistant to herbicides:
         Glyphosate (―Roundup‖) is a broad-spectrum
          herbicide that inhibits an enzyme system in
          chloroplasts that is involved in the synthesis of
          amino acids.
         A bacterial gene, which confers resistance to
          glyphosate, is inserted into useful food crops
          (corn, cotton, soybeans) to protect them from
          the herbicide, which otherwise would kill them
          along with the weeds.
16       Biotechnology: Applications of DNA Manipulation

     • Grains with improved nutritional characteristics:
         Genes from bacteria and daffodil plants are
          transferred to rice using the vector
          Agrobacterium tumefaciens.
         Now a genetically modified strain of rice
          produces b-carotene, a molecule that is
          converted to vitamin A in animals.
16       Biotechnology: Applications of DNA Manipulation

     • Crops that adapt to the environment:
         A gene was recently discovered in the thale
          cress (Arabidopsis thaliana) that allows it to
          thrive in salty soils.
         When this gene is added to tomato plants,
          they can grow in soils four times as salty as
          the normal lethal level.
         This finding raises the prospect of growing
          useful crops on previously unproductive soils
          with high salt concentration.
         Biotechnology may allow us to adapt plants to
          different environments.
16       Biotechnology: Applications of DNA Manipulation

     • There is public concern about biotechnology:
         Genetically modified E. coli might share their
          genes with the E. coli bacteria that live
          normally in the human intestines.
         Researchers now take precautions against
          this. For example, the strains of E. coli used in
          the lab have a number of mutations that make
          their survival in the human intestine
          impossible.
16       Biotechnology: Applications of DNA Manipulation

     • There are concerns that genetic manipulation
       interferes with nature, that genetically altered
       foods are unsafe, and that genetically altered
       plants might allow transgenes to escape to other
       species and thus threaten the environment.
     • Regarding safety for human consumption,
       advocates of genetic engineering note that
       typically only single genes specific for plant
       function are added.
     • As plant biotechnology moves from adding genes
       to improve plant growth to adding genes that
       affect human nutrition, such concerns will become
       more pressing.
16       Biotechnology: Applications of DNA Manipulation

     • The risks to the environment are more difficult to
       assess.
     • Transgenic plants undergo extensive field testing
       before they are approved for use, but the
       complexity of the biological world makes it
       impossible to predict all potential environmental
       effects of transgenic organisms.
     • Because of the potential benefits of agricultural
       biotechnology, most scientists believe we should
       proceed, but with caution.
16       Biotechnology: Applications of DNA Manipulation

     • With the exception of identical twins, each human
       being is genetically distinct from all other human
       beings.
     • Characterization of an individual by DNA base
       sequences is called DNA fingerprinting.
16       Biotechnology: Applications of DNA Manipulation

     • Scientists look for DNA sequences that are highly
       polymorphic.
     • Sequences called VNTRs (variable number of
       tandem repeats) are easily detectable if they are
       between two restriction enzyme recognition sites.
     • Different individuals have different numbers of
       repeats. Each gets two sequences of repeats, one
       from the mother and one from the father.
     • Using PCR and gel electrophoresis, patterns for
       each individual can be determined.
Figure 16.17 DNA Fingerprinting
16       Biotechnology: Applications of DNA Manipulation

     • The many applications of DNA fingerprinting
       include forensics and cases of contested
       paternity.
     • DNA from a single cell is sufficient to determine
       the DNA fingerprint because PCR can amplify a
       tiny amount of DNA in a few hours.
     • PCR is used in diagnosing infections in which the
       infectious agent is present in small amounts.
     • Genetic diseases such as sickle-cell anemia are
       now diagnosable before they manifest
       themselves.